Mechanistic interplay among the M184I HIV-1 reverse transcriptase mutant, the central polypurine tract, cellular dNTP concentrations and drug sensitivity

Abstract

We recently reported that the M184I 3TC resistant mutation reduces RT binding affinity to dNTP substrates. First, the HIV-1 M184I mutant vector displays reduced transduction efficiency compared to wild type (WT) RT vector, which could be rescued by both elevating the cellular dNTP concentration and incorporating WT RT molecules into the M184I vector particles. Second, the central polypurine tract (cPPT) mutation and M184I mutation additively reduced the vector transduction to almost undetectable levels, particularly in nondividing cells. Third, the M184I (-) cPPT vector became significantly more sensitive to 3TC than the M184I (+) cPPT vector, but not to AZT or Nevirapine in the dividing cells. Finally, this 3TC sensitizing effect of the cPPT inactivation of the M184I vector was reversed by elevating the dCTP level, but not by the other three dNTPs. These data support a mechanistic interaction between cPPT and M184I RT with respect to viral replication and sensitivity to 3TC.

Figure 1. Effect of cPPT mutation and cellular dNTP level on transduction efficiency of HIV-1 vector

(A) Nucleotide sequences of wild type and mutant NL4-3 cPPTs used in this study. The translated amino acid sequence of the cPPT is a part of HIV-1 integrase. (B) Effect of the cPPT mutation on the transduction of the WT RT vector in dividing (serum) and nondividing primary human lung fibroblasts (HLFs) with (serum starved with 1mM dN) and without (serum starved) the elevation of cellular dNTP levels. Primary HLFs were plated, 2.5×10⁵, in media containing 10% serum and then in media containing 0.2% serum for 48 hours in the presence and absence of 1mM dNs. HLFs cultured under these conditions were transduced with 2.77×10⁵ pg of WT RT HIV-1 vector with (+) or without (−) the cPPT. The transduced cells were analyzed by FACS for GFP expression at 48 hours post transduction and the transduction efficiency was plotted. The experiments presented in this figure were performed at least in triplicate.

Figure 2. Effect of cPPT mutation on the transduction efficiency and proviral DNA synthesis of HIV-1 vector harboring M184I mutant RTs

(A) Transduction efficiency of (+) and (−) cPPT M184I vectors in HLFs cultured in 10% serum, 0.2% serum and 0.2% serum with 1mM dNs. HLFs were transduced with an equal p24 level, 2.77×10⁵ pg of (+) and (−) cPPT M184I RT HIV-1 vectors, and the transduction efficiency was determined as described in Figure 1B. (B) Quantitative 2LTR circle PCR assay of the (+) and (−) cPPT M184I vector in the serum-starved HLFs. Genomic DNA of serum-starved HLFs transduced with the M184I virus was prepared at 48 hours post transduction and used for the quantitative 2LTR PCR to monitor the kinetics of the proviral DNA synthesis and normalized with genomic cellular DNA as previously described (Jamburuthugoda, Chugh, and Kim, 2006; Skasko and Kim, 2008). (C) The LTR/gag late reverse transcription product synthesized from the 3’ LTR was measured for the (+) and (−) M184I vector in the serum-starved HLF at 48 hours post transduction as described in Materials and Procedures. The treatment with 2µM Nevirapine and 1µM AZT, which block viral reverse transcription, was employed for assessing the PCR background control. The experiments presented in this figure were performed at least in triplicate.

Figure 3. Compensation of the transduction defect of the M184I (−) cPPT HIV-1 vector by the incorporation of WT HIV-1 RT

(A) (−) cPPT HIV-1 vectors containing different percentages of WT RT to M184I RT in a single viral particle (100% M184I, 90% M184I to 10% WT, 50% M184I to 50% WT, 10% M184I to 90% WT, 100% WT) were prepared. The serum-starved HLFs were transduced with an equal p24 amount of the constructed hybrid vectors. The pictures of the transduced cells in bright field and GFP were taken at 48 hours post transduction, and the overlaid pictures were constructed. At the same time, the transduction efficiency was determined by FACS analysis for the GFP-expressing cells (B). The experiments presented in this figure were performed at least in triplicate.

Figure 4. Effect of the cPPT mutation on 3TC sensitivity of HIV-1 M184I vector

(A) The transduction efficiency of the M184I (+) and (−) cPPT HIV-1 vectors in dividing HLFs in the presence of 50µM 3TC. (B) 3TC dose dependence of the M184I (+) and (−) cPPT vectors. (C) AZT dose dependence of the M184I (+) and (−) cPPT vectors. (D) Nevirapine dose dependence on the M184I (+) and (−) cPPT vectors. HLFs were transduced with an equal p24 level of the (+) and (−) cPPT vector in dividing HLFs pretreated with varying concentrations of 3TC, AZT or Nevirapine. The transduction efficiencies at each of the concentrations in (B), (C) and (D) were normalized with that of the drug free control (100%). The IC₂₅ and IC₅₀values were marked with a dotted line. The experiments presented in this figure were performed at least in triplicates.

Figure 5. Effect of dN treatment on M184I (−) HIV-1 vector sensitivity to 3TC

(A) 3TC dose dependence of the M184I (+) and (−) cPPT vectors in HLFs pretreated with 1mM dN. (B) The transduction of the M184I (−) cPPT vectors in dividing HLFs in the presence and absence of 1mM dC or a mixture of 1mM dA, dT, and dG. HLFs were transduced with an equal p24 level of the (−) cPPT M184I RT vectors in dividing HLFs pretreated with varying concentrations of 3TC. The transduction efficiency at each of the concentrations was normalized with that of the drug free control (100%). The experiments presented in this figure were performed at least in triplicate.